This invention relates generally to the suppression of bubble formation in a fluid having an interface with a gas. More particularly, the invention relates to a method and apparatus for containing a small quantity of fluid on a solid substrate while inhibiting bubble formation. A primary use of the invention is in solid phase chemical processes, e.g., in array hybridization.
Nucleic acid hybridization is a known method for identifying specific sequences of nucleic acids; hybridization involves base-pairing between complementary nucleic acid strands. When single-stranded nucleic acids are used as probes to identify specific target sequences of nucleic acids, probes of known sequences are exposed to and incubated in sample solutions containing sequences to be identified. If a sequence hybridizes to a probe of a known sequence, the sequence is necessarily the specific target sequence. Various aspects of this method have been studied in detail. In essence, all variations allow complementary base sequences to pair and thus form double-stranded stable molecules, and a variety of methods are known in the art to determine whether pairing has occurred, such as those described in U.S. Pat. No. 5,622,822 to Ekeze et al. and U.S. Pat. No. 5,256,535 to Ylikoski et al.
Hybridization of surface-bound probes to solution-based targets is an effective means to analyze a large number of DNA or RNA molecules in parallel. Specific probes of known sequences are attached to the surface of a solid substrate in known locations. The probes are usually immobilized on a solid support having a surface area of typically less than a few square centimeters. The solid support is typically a glass or fused silica slide which has been treated to facilitate attachment of probes. A mobile-phase sample containing labeled targets, e.g., a buffered aqueous solution containing target DNA, is contacted with and allowed to react with the surface. By detecting the labels to determine whether hybridization has occurred at specific locations, it is possible to determine the composition of the sample and the sequences of the unknown targets. Alternatively, target biomolecules may be bound to the surface while labeled probes are contained in the mobile phase. In either case, the hybridization reaction typically takes place over a time period that can be many hours, for a typical sample containing target material in the concentration range in the picomolar domain.
A number of factors contribute to the desirability of using samples that contain a small volume of fluid. First, as a general matter. the extent of the hybridization reaction is proportional to concentration. Since concentration is inversely proportional to volume, it is desirable to decrease the volume of fluid for any sample. In addition, where array technology is employed, it is now possible to attach an increasing number of distinct probes, each in a known location, on a relatively small substrate. Particularly to prevent waste of a limited supply of sample fluid, it is desirable to use only enough fluid to contact each probe feature of the array.
When an array is employed, every part of the probe-containing surface should have equivalent exposure to the target solution to ensure uniform hybridization conditions at each probe. Typically, an array is provided on a slide, and target solution is spread over the array with a cover slip. Only 10-20 xcexcl of sample fluid is needed to cover an array in the shape of a square with sides of 20 mm in length. The fluid layer is typically 25-50 xcexcm thick. In essence, the cover slip can be described as freely floating on the thinly spread solution and held in place only by the surface forces between the fluid and cover slip. The assembly is then placed in a humidified chamber which is in turn placed in an incubator at the desired temperature for hybridization. Hybridization is usually induced at elevated temperatures, and the humidity prevents the fluid layer from evaporating. While bubbles are generally not observed to form in the typical cover slip slide set-up, there are several problems with this assembly. The fluid layer is not always a constant thickness, resulting in more target molecules contacting some surface regions and fewer target molecules coming into contact with other surface regions. This is problematic because the extent of hybridization will vary across the surface. In addition, the assembly must be held flat and horizontal so that the cover slip does not slide off and uncover the array. Such assemblies are therefore difficult to handle and not easily adaptable to automated hybridization processes.
In the alternative, hybridizing arrays may be packaged in a sealed chamber such that sample fluid is placed in contact with the array within the chamber. Several packaging approaches have been demonstrated. U.S. patent application Ser. No. 09/299,976 describes an adjustable volume hybridization chamber. U.S. patent application Ser. No. 09/133,102 describes a centrifugal valve for containing and then controllably releasing the fluid sample. U.S. patent application Ser. No. 09/302,011 describes package solutions for annular format arrays. U.S. patent application Ser. No. 09/343,645 describes packaging solutions for multiple arrays and multiple packages. U.S. patent application Ser. No. 09/343,372 describes a reusable hybridization package. There are many advantages to packaging the arrays in sealed chambers. Since chambers containing the fluid are sealed, the risk of unacceptable fluid evaporation is eliminated or at least lowered. Packaging also allows the fluid height to be more controlled, thereby allowing for even hybridization. The ease with which packages can be handled, when compared with the floating cover slip assembly as described above, allows hybridization to be automated. In addition, packages may be designed such that the fluid is mixed within the package to ensure that no diffusion rate limited concentration gradient within the fluid develops during hybridization. The major disadvantage in using sealed packages is that bubbles tend to form in the hybridization fluids during the time it takes for hybridization to occur.
While bubbles may be employed to mix the hybridization fluid during hybridization, e.g., as described in U.S. patent application Ser. No. 09/137,963, bubbles are generally undesirable in hybridizations where a thin film of hybridization fluid is used, for a number of reasons. Bubbles are local inhomogeneities in the hybridization fluid. And if they remain substantially immobile during hybridization, the bubbles can result in uneven heat transfer and localized hot spots on the surface containing bound biomolecules. In an extreme case, bubbles may disrupt the continuity interface between the hybridization fluid and the surface containing bound probes, thereby displacing fluid away from surface-bound probes and preventing target species from contact with the probes.
There have been a number of means developed for minimizing the adverse impact of bubble formation. For example, a surfactant may be added to the hybridization solution and the solution continuously mixed to ensure that the bubbles are mobile and prevented from remaining at any particular location. In addition, U.S. Pat. No. 5,959,098 to Goldberg et al., U.S. Pat. No. 5,945,334 to Besemer et al. and U.S. Pat. No. 5,922,591 to Anderson et al. each describe other bubble management means such as employing nonparallel top and bottom surfaces in a hybridization chamber to reduce the potential of trapping the bubbles. Another such bubble management means involves moving the hybridization solution in and out of the chamber throughout the hybridization process. All these references address the problems associated with bubble formation, but do not provide a means for inhibiting bubble formation.
Thus, there is a need for a method and apparatus to inhibit bubble formation in a hybridization package adaptable for automated processing.
Accordingly, it is an object of the present invention to overcome the above-mentioned disadvantages of the prior art by providing a new and effective apparatus for inhibiting bubble formation during a chemical reaction.
It is another object of the invention to provide such an apparatus which is capable of carrying out the chemical reaction with a very small amount of fluid.
It is still another object of the invention to provide such an apparatus wherein the chemical reaction involves the hybridization of biomolecules.
It is a further object of the invention to provide a new and effective method for inhibiting bubble formation during a chemical reaction conducted on a solid surface.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.
In one aspect, then, the invention relates to an apparatus for inhibiting bubble formation during a chemical reaction. The apparatus comprises a base typically having a substantially planar surface with at least a portion of the surface representing a fluid contact area and a fluid comprising a liquid component in contact therewith. A cover which sealingly contacts the base directly or indirectly about the fluid contact area forms an enclosure containing the fluid and a gas. Attached to the cover or the base is a fluid-distribution member having preferably a substantially flat surface in contact with the fluid. The member surface is disposed in an opposing manner, ideally also substantially parallel manner, at a specified distance from fluid contact area. A gas-fluid interface having an interface radius is formed between the fluid and the gas. The apparatus also comprises means for maintaining a desired vapor pressure of the liquid component in the gas and means for immobilizing the cover with respect to the base. The interface radius is selected to result in a predetermined critical radius below which a bubble will shrink. The predetermined critical radius should be equal to at least half the distance between the member surface and the base surface.
In another aspect, the invention relates to the apparatus as above wherein the fluid comprises a biomolecule. The biomolecule may be an oligonucleotide, a polynucleotide, an oligopeptide or a polypeptide. The biomolecule may be adapted to react with an array of features attached to the fluid contact area.
In another aspect, the invention relates to the apparatus as above wherein the means for maintaining the desired vapor pressure comprises a supply of the liquid component independent from the fluid in vapor communication with the interface between the fluid and gas. The supply of the liquid component may comprise the liquid component contained in a compartment located within the enclosure. In addition, the desired vapor pressure may correspond to a condition where liquid component is nearly or fully saturated in the gas. For example, the liquid component may be water and the condition may be no less than about 90% rH.
In a further aspect, the invention relates to the apparatus as above further comprising a sealable fill conduit that extends through the cover. The conduit terminates in an outlet within the enclosure. The outlet of the conduit may be substantially centrally located on the fluid-distribution member. The apparatus may further comprise a vent port located on the cover.
In still a further aspect, the invention relates to the apparatus as above wherein the fluid-distribution member comprises a plurality of openings located on the member surface to provide fluid with vapor communication with the gas to form the gas-fluid interface. The fluid-distribution member may comprise a screen. In addition, the member surface or the base surface may comprise a hydrophobic material such as a fluorinated or perfluorinated polymeric substance.
In another aspect, the invention relates to a method for inhibiting bubble formation in a chemical or a biochemical reaction. To carry out the method the following are provided: a base having a substantially planar surface representing a fluid contact area; a cover capable of sealingly contacting. the base directly or indirectly about the fluid contact area; and a fluid distribution member having a substantially flat surface. Fluid comprising a liquid component is dispensed on the fluid contact area. A cover is placed in direct or indirect sealing contact with the base to form an enclosure. A desired vapor pressure of the liquid component in the gas is effected in the enclosure. The member surface is maintained in non-free-floating contact with the fluid such that the member surface and the base surface are disposed at a specified distance of each other and in an opposing and substantially parallel manner. As a result, a gas-fluid interface is formed having a gas-fluid radius that is selected to provide a predetermined radius below which a bubble will shrink.